The objectives of this study were

The objectives of this study were to investigate 1) the mRNA and protein expression of apelin and APJ in porcine ovarian follicles of different sizes, and their immunolocalization and concentrations in follicular fluid and ovarian follicle, and 2) the direct effect of recombinant human apelin-13 on basal and IGF1- and FSH-stimulated steroidogenesis and cell proliferation in co-culture of granulosa (Gc) and theca (Tc) cells. As a molecular mechanism of apelin action on ovarian steroidogenesis, we propose the activation of adenosine 5′-monophosphate-activated protein kinase (AMPK), and the promotion of cell proliferation by the activation of phosphatidylinositol 3′-kinase/Akt and the extracellular-signal-regulated kinase (ERK1/2) signaling pathway. Studies have provided evidence that AMPK is involved in the regulation of ovarian steroid production in various species [25], [26], [27], while ERK1/2 and Akt signaling pathways are widely expressed in ovarian 97 9 and are involved in cell survival and proliferation in the ovary [28], [29], [30].
Material and methods
Results
Discussion This study confirms for the first time that apelin and APJ are expressed in porcine ovarian follicles. Protein and mRNA levels change during follicular growth, with the highest expression in LFs. Our results are in agreement with Shimizu et al. [21] who observed that in bovine ovary theca cells, APJ mRNA expression increases with follicle growth. The differences in the ovarian expression of apelin and APJ (in follicles of different sizes) appear to be associated with hormonal regulation and the effect of endogenous hormones, the extent of which change during the ovarian cycle. Follicular dynamics is characterized by increasing concentrations of estradiol, driven by increasing concentrations of pituitary gonadotropins. The gonadotrophins are, in turn, regulated by gonadotropin-releasing hormone from hypothalamic neurons [38]. This assumption is supported by the work of Shimizu et al. [20], who showed that the mRNA level of APJ in bovine granulosa cells treated with P4 and E2 increases. Other studies have reported that mRNA expression of apelin is regulated by several different factors such as LH [20], IGF1 [24] and insulin [2], [39]. Additionally, ovarian expression of other adipokines, such as resistin, is known to increase using steroid hormone and gonadotropins [40]. The findings of this study are in agreement with other research that documented the expression of both components (ligand and receptor) of the apelin signaling system in ovarian cells collected from bovine [19], [20], [21], rats [15], [17] and humans [24]. Here, we have demonstrated that apelin and APJ are immunolocalized in membranes of granulosa cells, than theca. A strong positive immunoreaction of apelin was observed in the oocyte, whereas the reaction of APJ was stronger at the zona pellucida. In human ovaries, both apelin and its receptor are localized in different compartments such as granulosa, oocyte, cumulus and theca cells [24], while in bovine ovaries, APJ is expressed in the granulosa cells of follicles at different developmental stages. However, both apelin and APJ are present in theca cells of bovine ovaries [20]. Overall, the expression of both components of the apelin signaling system provides the opportunity for an analysis of the role of apelin in porcine ovarian follicles. The in vitro effects of recombinant apelin on steroid hormone secretion and steroid enzyme expression may represent a unique mechanism of apelin action in mature animals. The recombinant apelin increased P4 and E2 secretion, which was confirmed by additional experiments on the effect of apelin on the steady state levels of 3βHSD, and CYP19 mRNA and protein expression. These findings are also consistent with a recent in vitro study by Roche et al. [24] who showed that apelin increases steroid production and 3βHSD protein basal levels, and in response to IGF1 in human granulosa cells. However, in this study, apelin induced by IGF1 and FSH led to a decrease in steroid secretion and steroid enzyme protein expression. This suggests that the differences in the effect of apelin on IGF1 and FSH–induced steroid production is associated with the ovarian cell culture model employed. The primary co-culture models of theca and granulosa cells used in this study were from medium sized follicles of slaughterhouse pigs. In contrast, Roche et al. [24] used human granulosa cells obtained from pre-ovulatory follicles from women undergoing in vitro fertilization after hormonal stimulation. Hormonal homeostasis has a fundamental importance in the normal functioning of the ovarian follicles. Gonadotropins play primary roles in the initiation and maintenance of follicular growth, as well as in the selection of the dominant follicle and its maturation to preovulatory status. The developing responsiveness of follicles to stimulation by gonadotropins may result from changes in the production and alterations in follicular sensitivity to intraovarian paracrine and/or autocrine factors such as IGF1 and steroid hormones. IGF1 is one of several growth factors that play important roles in the regulation of follicular cell steroidogenesis especially as proliferation depends on follicular size [41]. Inhibitory effects of other adipokines such as adiponectin, leptin, resistin or chemerin, on steroid secretion induced by IGF1 or FSH, have been demonstrated in the ovary [10], [11], [40]. This study has demonstrated protein expression and immunolocalization of AMPKα in porcine ovary for the first time. High levels of AMPKα in oocyte and granulosa cells of LFs were observed, although the lower levels measured in theca cells is in agreement with a previous study of rat and bovine species [25]. In rat ovary, AMPKα was immunolocalized in oocytes and theca cells, although less abundantly than in granulosa cells [25]. However, Santiquet et al. [26] claimed that AMPK has an important role in porcine ovarian physiology, especially oocyte maturation. Moreover, in this study recombinant apelin was shown to increase the phosphorylation of AMPKα after 15 min of incubation. Using potent antagonists of APJ and AMPKα, a molecular mechanism of apelin action in the ovarian steroidogenesis can be described as follows: APJ is involved in the action of apelin on both P4 and E2 secretion in ovarian steroidogenesis, while AMPKα is only important in P4 secretion.